Genetic defects which cause diseases such as cancer, cystic fibrosis, muscular dystrophy and many other disorders contribute greatly to the cost of human health care. Consequently, there are major research efforts worldwide to develop new approaches to provide genetic therapy effective at the level of the somatic cell.
Central to this work are a variety of gene vectors derived from viruses such as retro-, pox, adeno-associated and adenoviruses. The viruses from which these vectors are derived are of low pathogenicity and the vectors are designed to carry therapeutic genes into the host cell. Under appropriate conditions these may produce products which can vaccinate the host, change cell phenotypes, stimulate immune responses, supplement genetic defects or lead to cell death.
No single viral vector has all the attributes desirable for all therapeutic situations. Some vectors are better suited to particular tasks than others because of their biological properties. For example, as retroviruses can integrate into the host genome, these vectors appear to be well suited to deliver genes to dividing cells such as the stem cells of the haemopoietic cell lineage. In contrast, adenovirus vectors do not usually integrate their DNA but are able to efficiently infect non-dividing cells. Human adenoviral vectors have been used to deliver genes to a variety of tissues in humans and in animal models, eg, lungs, muscle, liver, vascular and brain cells. A major disadvantage of the use of human adenoviruses is that as these viruses naturally infect humans, it can often be the case that the person to be treated with the human adenoviral vector has circulating antibodies which can neutralise the vector prior to it reaching the target cell. Immunity also results in efficient clearance of the virus from the host by the cellular immune system, thereby rapidly diminishing any therapeutic effect due to gene expression. Thus, there is a need to develop vectors derived from viruses that normally do not infect humans, so as to overcome the problem of pre-existing immunity. As ovine and human adenoviruses are serologically distinct, the use of an ovine adenovirus (OAV) as a vector may achieve this objective. It is also essential to develop vectors whose presence in the host is disguised so that the immune response is less severe and gene expression is more likely to persist. As ovine adenovirus does not replicate productively, even in most non-ovine animal cells (1) it was expected to replicate abortively in human cells, provided that it could infect them. A block in replication at all early step would lead to minimal expression of viral products in the infected cell, with a consequent low level induction of the cellular immune response.
The present inventors have developed an adenoviral vector derived from ovine adenovirus OAV287. The virus and viral vectors derived therefrom are fully described in International Patent Application Number WO 96/03508 filed on Jul. 26, 1995 in the name of Commonwealth Scientific and Industrial Research Organisation (hereinafter referred to as "the PCT Application" and incorporated herein by reference). The adenoviral vector disclosed in the PCT Application was found to be suitable for use as a vector to introduce foreign DNA into a variety of non-human cells, and in particular into sheep cells. The genome sequence and arrangement of OAV287 is different from all known human, animal and avian adenoviruses, including the canine adenovirus described in International Patent Application Numbers WO 91/11525 and WO 94/26914, the bovine adenovirus described in International Patent Application Number WO 95/16048 and the avian CELO isolate (2). OAV is also serotypically distinct (1) and is not neutralised by serum human Ad5. This is consistent with the distinctive amino acid sequences in the hexon, penton and fiber antigens (3). There are also other major differences in the capsid proteins of OAV compared with other known adenoviruses. OAV lacks capsid protein homologues V and IX but contains at least two other structural proteins (3). Due to the low nucleotide sequence homology between OAV and other adenoviruses there is little chance of recombination between OAV and another adenovirus during co-infection in the host to form an infectious recombinant virus.
Critical to the use of OAV vectors for human gene therapy is the question of whether OAV call actually enter human cells. Entry of human adenoviruses into human cells occurs via a two step process involving specific amino acid sequences in the fiber and penton base proteins (4, 5). First the virus attaches to an unidentified surface receptor via the trimeric fiber protein which protrudes from the surface of the virus. Secondly, an interaction occurs (primarily) between the .alpha..sub.v .beta..sub.v class of integrins and an Arg/Gly/Asp (RGD) tri-peptide which forms part of the penton protein complex at the base of the fiber spike (6). The virus is then taken into the cell by endocytosis. The cell binding domain of the ovine adenovirus OAV287 fiber protein is smaller and has a completely different sequence to its human adenovirus homologues. It almost certainly binds to a different primary receptor. Compared with human adenoviruses, the OAV penton protein also lacks the critical RGD motif and flanking sequences (3). Thus, it was not known, nor could it be predicted. whether OAV including OAV287 would infect human cells.
The present inventors have made the surprising discovery that, despite the major differences in its cell attachment proteins, viral vectors derived from ovine adenovirus OAV287 can infect a variety of, but not all, human cell lines. This finding paves the way for the use of OAV as a human gene therapy vector.